Refined understanding of the impact of the Mycobacterium tuberculosis complex diversity on the intrinsic susceptibility to pretomanid

ABSTRACT Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1–9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2–4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2–4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT. IMPORTANCE This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Second, clinical strains with high Pa MICs due to mutations in known Pa resistance genes were identified without known nitroimidazole exposure, suggesting genetic drift or yet unknown selective pressures (3)(4)(5).In the few settings with good surveillance or routine antimicrobial susceptibility (AST) results, these mutants are rare (5,6).However, because these mutants are known to be transmissible, it is plausible that some settings exist in which an intrinsically Pa-resistant cluster is frequent, underlining the need for routine AST (4,7).Yet, rapid AST directly from clinical samples is currently impossible as no commercial genotypic AST assay exists that interrogates ddn (Rv3547), fbiA (Rv3261), fbiB (Rv3262), fbiC (Rv1173), fbiD (Rv2983), and fgd1 (Rv0407), the six genes required for the activation of the pro-drug Pa [no resistance mutations have been described in dprE2 (Rv3791)], the target of Pa, to date) (8)(9)(10).Although efforts are underway to address this diagnostic gap (e.g., Genoscreen is evaluating Deeplex Myc-TB XL, an updated version of its WHO-endorsed targeted next-generation sequencing assay), the interpretation of genotypic AST results will remain a persistent challenge as the aforementioned resistance genes are non-essential and, consequently, thousands of different loss-of-function mutations can theoretically confer resistance (9,11,12).
The goal of this study was twofold.First, we used MGIT to refine the current under standing of the effect of the MTBC diversity on susceptibility, with a particular focus on the less frequent lineage 5 (L5), lineage 6 (L6), lineage 7 (L7), lineage 8 (L8), and lineage 9 (L9) that were not tested or were underrepresented in the literature (3,13,14).Second, we used Middlebrook 7H11 (7H11) as an alternative medium to investigate whether the differences observed with MGIT were media specific.

Strains
We tested 125 MTBC strains from L1-9 from patients who had never received nitroimi dazole treatment originating from 45 different countries on five different continents.Of these, 118 lacked known resistance mutations in the six canonical Pa-resistance genes and the remaining seven did not have whole-genome sequencing data but were selected based only on the treatment naivety to nitroimidazoles to augment L5 and L6.Of 125 strains, 49% (n = 61) were drug-susceptible (DS), 27% (n = 34) were mono-/ poly-resistant (mono/PDR) to other TB drugs other than Pa, 23% (n = 29) were multidrugresistant (MDR), and 1% (n = 1) was pre-extensively drug-resistant (Table S1) (15).All 125 strains were tested on 7H11, whereas a subset of 41 isolates were tested in MGIT.In addition, four Pa-resistant strains with known resistance mutations were included for both methods (Table 1).Of the total 129 strains, 10 strains were also tested by Bateson et al. (Table S2) (3).As per the ITM-IRB consultation, the fully anonymized use of clinical isolates for test validation did not require ethical review.
A twofold dilution series of Pa ranging from 1 to 0.002 µg/mL plus 0.75 µg/mL (i.e., 11 concentrations in total) were tested for all strains, except for the four Pa-resistant strains, for which 0.25-8 µg/mL were used instead.Bacterial colonies were scraped from Löwenstein-Jensen slants and thoroughly homogenized in sterile water with glass beads.The density of the suspension was adjusted visually to McFarland 1.The least diluted growth control (GC1) and the drug-containing media in polypropylene tubes were inoculated with a 10 −1 dilution of the McFarland 1 suspension, while the most diluted growth control (GC2) was inoculated with a 10 −3 dilution.Colony-forming units (CFU) were enumerated after 4 weeks of incubation at 34°C-38°C with 5%-10% CO 2 .If both growth controls had sufficient growth at this point [i.e., at least 1+ (51-100 CFUs) on GC1 and 3 CFUs on GC2], CFU counts were recorded accordingly, and MIC results were interpreted.If GC1 and/or GC2 had insufficient CFUs at 4 weeks, tubes were incubated for two more weeks.Any test with insufficient CFUs on GC1 and/or GC2 after 6 weeks of incubation or more than 1+ growth on the GC2 was considered invalid and repeated once.The MIC was defined as the lowest drug concentration that inhibits the growth of more than 99% of the MTBC population.
Since MIC testing of Pa on 7H11 had not yet been established in our laboratory at the Unit of Mycobacteriology, Institute of Tropical Medicine, we first tested 30 replicates of the pan-susceptible H37Rv reference strain (BCCM/ITM CT2008-03715/ITM500735), using three different batches of Pa-containing medium, with repeated testing on different days over 10 weeks.Subsequently, H37Rv was included as a control in every batch of clinical strains.

MGIT MIC testing
Pa working solutions prepared from the same stock solution used for 7H11 testing were added to MGIT tubes (100 µL each) to achieve 10 twofold Pa dilutions from 0.002 to 1 µg/mL, whereas higher concentrations (0.25-4 µg/mL) were tested for Pa-resistant strains.An inoculum was prepared directly from a positive MGIT tube that had flagged within 1-2 days or after a one in five dilution of a positive MGIT tube that had flag ged within 3-5 days, and 500 µL of inoculum was added to the Pa-containing tubes supplemented with 800 µL of OADC.The drug-free control vial was inoculated with a 1:100 dilution of the inoculum.MICs were determined using MGIT 960 TBeXIST extended protocol according to the manufacturer's instructions.The MIC was determined to be the lowest concentration at which the growth value of the drug-containing tube was <100 when the growth control had reached 400 growth units.A test resulting in an invalid code (×200 or ×400) was repeated once.Based on Bateson et al. (3), 0.06-0.5 µg/mL was used as a tentative quality control (QC) range, and a corresponding QC target of 0.125-0.25 µg/mL for H37Rv, which was included in every batch of clinical strains.

Technical reproducibility of Pa MIC testing
A good technical reproducibility was observed for both methods.Indeed, Pa MICs in MGIT were 0.125-0.25 µg/mL for H37Rv, corresponding to the tentative QC target (Table 2) (3).The corresponding 7H11 MICs were 0.06-0.125µg/mL (no tentative QC range/ target was available for comparison).Two strains were tested in both studies (Table S2).
b Three strains were tested in both studies (Table S2). c One strain was tested in both studies (Table S2).g

MICs for strains with Pa resistance mutations
The four strains with known Pa resistance mutations had MICs of >4 µg/mL in MGIT, which was in line with earlier results, and >8 µg/mL using 7H11 (Table 1) (3).

MICs for strains without Pa resistance mutations
Bateson et al. ( 3) reported a mode of 1 µg/mL and 99th percentile of 2 µg/mL for L1 MICs in MGIT, which was elevated compared with the mode of 0.125 µg/mL and 99th percentile of 0.5 µg/mL for L2-4/7.The MGIT MIC data from this study agreed with these findings (Table 2).L6 strains were even more susceptible with MICs ≤ 0.016 µg/mL in both studies.L5 MICs were 0.03-0.06µg/mL in both studies, but we tested a greater number of strains (eight vs just two in the literature), demonstrating that the susceptibil ity of L5 was more similar to that of L2-4/7 than that of L6.Based on a single replicate for one strain each, it was unclear whether L8 was more similar to L1 or L2-4/7, whereas L9 most resembled L6.Similar relative susceptibilities of the different lineages were observed on 7H11 (Table 2).For example, the 99th percentile of the L1 distribution at 1 µg/mL was two doubling dilutions higher than for L2-4.MGIT MICs for L1-4 were approximately twice as high as the corresponding 7H11 MICs (Table 3), which was also apparent when comparing the modes of their MIC distributions (Table 2).In contrast, the absolute MICs for L5 and L6 were similar for both media.As only a single strain was tested for L7-8, no meaningful comparison was possible for these lineages.

DISCUSSION
This study confirmed that Pa MICs were elevated in L1, regardless of the medium used and phenotype measured [7H11 MIC testing relies on visual growth inhibition on solid medium, whereas MGIT measures oxygen consumption in liquid medium (16)].The fundamental question for regulators is whether BPaL(M) should be used for L1, even though the clinical outcome data demonstrating good outcomes are more limited for L1 compared with L2-4, therefore, remains pressing in light of the ongoing adoption of this regimen globally (3).In January 2023, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) set a "provisional screen value" of 2 µg/mL for MGIT, which was reaffirmed in 2024, without an accompanying explanation of the meaning or intended use of this concentration (17,18).Given a history of mistakes when setting breakpoints for MTBC by multiple regulators, we call on EUCAST to publish a justification for its decision to enable external scrutiny (19)(20)(21)(22).Moreover, EUCAST should engage with the European Medicines Agency to review its breakpoint for MGIT given that the current choice of 1 mg/L for MGIT is too high for L2-4/7 and low high for L1 (23).WHO reviewed these questions independently and is due to publish its decision shortly.
a L9 was excluded as no 7H11 MIC was available, and the ratio was not calculated for strains with at least one truncated MIC (see Table S2 for more details).
L6, which causes up to half of TB in some West African countries yet appears underrepresented among rifampicin-resistant strains, was more susceptible than L5 and L2-4/7 and should, therefore, respond better to BPaL(M) (3,24).More MICs are needed for L8, L9, and the different animal-adapted MTBC genotypes (e.g., Mycobacterium bovis), and it would be desirable to clarify their likely response to BPaL(M), although this is not a priority as these are much rarer than L1-7 (3,14,24).
MICs for L1-4 appeared to be systematically lower in 7H11 than in MGIT in this single-site study, requiring confirmation in other laboratories [i.e., technical variability may account for this apparent difference, as previously observed for H37Rv tested in different media in MGIT (3,(25)(26)(27)].
Our findings further underline the importance of the EUCAST requirements to consider MIC data from multiple laboratories and from phylogenetically diverse MTBC strains (3,25,26).Accordingly, the TB Alliance is preparing a study to define the L1 and non-L1 MIC distributions using the EUCAST reference method (26).Commercial phenotypic AST devices (e.g., a lyophilized Pa product for MGIT or a lyophilized broth microdilution assay, which would be preferable to manually weigh Pa for MGIT testing as is the only option currently) will have to be calibrated against the reference method to ensure that any MIC differences are fully systematic (e.g., that the technical variability is not excessive, resulting in wider MIC distributions and, thus, increasing the likelihood of very major diagnostic errors) (26,28,29).In the future, such quality-assured and comprehensively validated commercial AST assays should be co-developed with novel relevant antimicrobials given that empiric use risks their long-term utility (30)(31)(32).

TABLE 2 1 (TABLE 2
MICs for H37Rv and clinical strains without known Pa resistance mutations g Continued on next page) MICs for H37Rv and clinical strains without known Pa resistance mutations g

TABLE 1 MICs for Pa-resistant strains d Strain ID Lineage DR profile Genome accession Pa resistance mutation Study Pa MIC (µg/mL)
(12) vitro mutant.bSelectedby WHO as a resistant control strain for delamanid and Pa in the forthcoming AST manual.cRecognizedas conferring cross-resistance to delamanid and Pa in the second edition of the WHO mutation catalogue(12).

TABLE 3
MGIT to 7H11 MIC ratio for H37Rv and clinical strains without Pa resistance mutations a